According to data from the Sociedade Brasileira para o Estudo da Dor [Brazilian Society for the Study of Pain] (SBED, 2004 http://www.dor.org.br/dor_impactos.asp), pain affects at least 30% of individuals at some moment of their lives and, from 10% to 40% of these individuals, pain lasts for more than one day. Pain is the main cause of suffering, work incapacity and it provokes serious psychosocial and economical consequences. Approximately 40% of these individuals miss many days of work. There are no official statistics about the impact of pain conditions in Brazilian population, however its occurrence has increased substantially in the last years. In addition, the incidence of chronic pain in the world oscillates from 7% and 40% of the population. As a consequence, from 50% to 60% of those individuals that suffer from chronic pain become partially or totally, temporarily or permanently incapacitated, significantly compromising life quality.
The therapeutic use of snake venoms observed in humans dates back to the beginning of the 20th century (Brasil, V. Biol. Med. São Paulo, 1: 7-21, 1934, Brazil, V. An. Paul. Med. Cir., 60: 398-408, 1950; Klobusitzky D. Anais do Instituto Pinheiros, 1:3-23, 1938) and literature presents important reviews of the use of these venoms as therapeutic agents. These reviews show, for example, the use of the venom of Crotalus adamanteus for the treatment of epilepsy and the use of the venom from Agkistrodon piscivorus, Vipera ruselli and Notechis scutatus as haemostatic agents (Klobusitzky, D. Anais do Instituto Pinheiros, 1:3-23, 1938).
Reports about the analgesic property of snake venoms observed in humans date back to the beginning of the 30's decade (Monaelesser & Taguet, 1933, apud on Brazil, V. An. Paul. Med. Cir., 60: 398-408, 1950). Concerning the analgesic effect of the venom of the South American rattlesnake (Crotalus durissus terrificus), hereinafter referred to as “CdtV”, the first studies were performed by Dr. Vital Brazil. In these studies, Dr. Vital Brazil prepared highly diluted crotalid venom solutions, denominated crotalid solute. The crotalid solution was distributed to several physicians in Brazil and abroad and it was used for the treatment of different pain conditions and disorders, mainly of neoplasic origin. The results of this study demonstrated that the rattlesnake's venom is highly effective in the treatment of different pain syndromes (Brazil, V. Biol. Med. São Paulo, 1: 7-21, 1934, Brazil, V. An. Paul. Med. Cir., 60: 398-408, 1950).
Concerning the use of snake venom-derived products for the treatment of painful conditions, the development of a product called anavenom, produced at Institute Butantan, by the mixture of these venoms with formaldehyde, is worth highlighting. These products were indicated for the treatment of different painful conditions, particularly in cases where usual analgesics had no effect. This product demonstrated potent analgesic effect, since this product could substitute the treatment with morphine. The anavenom also demonstrated long lasting analgesic effect, as patients were usually treated with anavenom with 1 to 3 days intervals between doses.
Despite the results observed by Dr. Vital Brazil, showing the analgesic effect of Crotalus durissus terrificus snake venom, the active substance, present in the crude venom, responsible for the analgesic effect, was not known.
Studies of the mechanisms of the analgesic action of this venom, using experimental models of pain evaluation, began in 1990.
These studies showed that CdtV, administered in mice, induces a long lasting antinociceptive effect, when evaluated in the hot plate test, suggesting that this venom is capable of causing analgesia through an action in the Central Nervous System (Giorgi R. et al., Toxicon, 31: 1257-65, 1993; Picolo G. et al. Toxicon 36:223-227, 1998). Pharmacological studies showed the involvement of kappa opioid receptors (Giorgi R. et al., Toxicon, 31: 1257-65, 1993; Brigatte P. et al. Toxicon 39:1399-1410, 2001), in antinociception observed in the hot plate test. Long treatments using the venom induced tolerance to the antinociceptive effect in the hot plate test, but not physical dependence. Tolerance is mediated by pharmacodynamic mechanisms. Crossed tolerance with morphine was not observed (Brigatte P. et al. Toxicon 39:1399-1410, 2001). On the other hand, due to the long lasting antinociceptive effect of the venom (5 days after the administration of a single dose), there is no development of the tolerance phenomenon if the venom is administered every 5 days, for up to 65 days after the beginning of the treatment (Brigatte P. et al. Toxicon 39:1399-1410, 2001).
In addition to the effect observed in the hot plate test, analgesic action was demonstrated for the crude venom in two experimental models of inflammatory pain: the model of the abdominal contortions induced by acetic acid (Giorgi R. et al., Toxicon, 31: 1257-65, 1993) and in hyperalgesia induced by carrageenin (Picolo G. et al., Eur J Pharmacol 391:55-62, 2000). In the carrageenin model, the analgesic effect of the venom is also a long lasting effect, persisting up to 5 days after the administration of one single dose of the venom. This effect involves the participation of peripheral delta opioid receptors (Picolo G. et al., Eur J Pharmacol 391:55-62, 2000).
On the other hand, in the model of hyperalgesia induced by prostaglandin, the antinociceptive action of the crotalid venom is mediated by kappa and delta opioid receptors (Picolo G. et al. Eur J Pharmacol 469:57-64, 2003). In both models of hyperalgesia (carrageenin and prostaglandin), the antinociception induced by the CdtV also involves the stimulation of the L-arginine/Nitric Oxide (NO)/cGMP pathway, of the cGMP-dependent protein kinase and activation of ATP-sensitive potassium channels (Picolo G. et al. Eur J Pharmacol 469:57-64, 2003, Picolo and Cury, Life Science 75:559-73, 2004).
It is important to emphasize that the venom is also able to induce antinociception in models of persistent pain, as in the model of neuropathic pain induced by chronic constriction of the sciatic nerve of rats (Gutierrez, V. P., Chacur, M., Sampaio, S. C., Picolo, G., Cury, Y. Memórias do Instituto Butantan vol. 60, p. 50, 2003) and in the model of cancer pain induced by the intraplantar injection of Walker 256 carcinoma cells in rats (Brigatte, P., Sampaio S. C., Gutierrez, V., Curi, R. Rangel-Santos, A. C., Guerra, J. L., Cury Y., XXXVI Congresso Brasileiro de Farmacologia e Terapêutica Experimental, Programa e Resumos, p. 195, 2004). The venom effect in the neuropathic pain model is also long, as it was detected for up to 3 days after the administration of a single dose of the venom. As observed in the models of hyperalgesia induced by carrageenin or prostaglandin, kappa and delta opioid receptors, the L-arginine/NO/cGMP pathway and opening of ATP-sensitive potassium channels are responsible for the effect of the venom in this model (Gutierrez, V. P., Chacur, M., Sampaio, S. C., Picolo, G., Cury, Y. Memórias do Instituto Butantan, vol. 60, p. 50, 2003).
Snake venoms are constituted by a complex mixture of proteins and biologically active peptides. Several active substances with different therapeutic indications were already isolated from these venoms. As examples, we have the patent U.S. Pat. No. 5,182,260 (Maraganore, J. H., 1993), where a polypeptide inhibitor of platelet activation was isolated from the North American Water Moccasin snake venom, or the patent U.S. Pat. No. 5,763,403 (Lyan, E. C. Y., 1998), where a lupus anticoagulant protein was obtained from the venom of Agkistrodon halys brevicaudus snakes, or the patent U.S. Pat. No. 6,489,451 (Li, B. X., 2002), where an antithrombotic enzyme was purified from the venom of Agkistrodon acutus snakes. In relation to products used in the treatment of pain, the American patent U.S. Pat. No. 6,555,109 (Shulov, A., 2003) describes a non-toxic fraction, isolated from the venom of Vipera xanthina palestinae snakes, and its derived products used to control diverse types of pain, including chronic pain. Furthermore, the American patent U.S. Pat. No. 6,613,745 (Gopalakrishnakone, P., 2003) presents peptides with sequences of amino acids derived or based on the amino acid sequence of an analgesic factor present in the venom of the King Cobra (Ophiophagus hannah).
The analgesic activity of crotamine, a toxin present in the venom of Crotalus durissus terrificus snakes, is also found in the literature. Studies demonstrated that there is an analgesic dose-response relationship, when purified crotamine is injected by s.c. or i.p. routes in mice. The analgesic effect was inhibited by naloxone, suggesting the involvement of opioid receptors (Mancin C. A. et al., Toxin 36:12, 1927-1937, 1998).
Opium and its derived products are potent analgesics, which also have other pharmacological effects. The endogenous and exogenous opioids are among the most used analgesics for pain control, particularly chronic or intractable pain, for example, cancer pain, neuropathic pain and chronic inflammatory pain. These drugs, by acting on specific receptors, induce analgesia in human beings and in animals, modifying the pathophysiological reponse to noxious chemical, mechanical or thermal stimuli (Yaksh, T. L. Acta Anaesth. Scand. 41:94-111, 1997).
At least three distinct families of endogenous opioid peptides were identified: the enkephalins, the endorphins and the dynorphins. Each family is derived from a distinct polypeptide precursor and has a characteristic anatomical distribution. These precursors, denominated proenkephalin, proopiomelanocortin and prodynorphin, have the Tyr-Gly-Gly-Phe-Met/Leu amino acid sequence (where Tyr, Gly, Phe, Met and Leu correspond to the tyrosine, glycine, phenylalanine, methionine and leucine amino acids, respectively), located in the N-terminal portion of the opioid peptides (Przewlocki R. and Przewlocka B. Eur. J. Pharmacol. 429:79-91, 2001, Reisine T. and Pasternak, G. In: The Pharmacolcogical Basis of Therapeutics, Hardman J. G. e Limbird L. E. eds, 9th ed, New York, McGraw-Hill, pp. 521-555, 1996).
The opioids, by acting on afferent nerve endings, inhibit adenylyl cyclase, decreasing the production of cAMP (Schultz, J. E. J. and Gross, G. J. Pharmacol. Ther., 89:123-137, 2001) and inhibiting the opening of calcium channels, with consequent blocking of the release of neurotransmitters (Junien, J. L. and Wettstein, J. G. Life Science, 51:2009-18, 1992; Zaki, P. A. et al. Annu. Rev. Pharmacol. Toxicol., 36:379-401, 1996; Yaksh, T. L. Acta Anaesth. Scand. 41:94-111, 1997).
In addition, opioids activate the L-arginine-nitric oxide-GMPc pathway, inducing potassium channel opening, with consequent hyperpolarization of the cellular membrane (Ferreira, S. H. et al. Eur. J. Pharmacol., 1217: 225-7, 1991; Ferreira, S. H. et al. Br. J. Pharmacol., 114: 303-8, 1995; Nozaki-Taguchi, N. and Yamamoto, T. Anesth. Anal., 87: 388-93, 1998; Amarante, L. H and Duarte, I. D. Eur J Pharmacol., 454:19-23, 2002). The antinociceptive effect of opioid agonists on K+ channels involves both the ATP sensitive and the voltage-dependent K+ channels (Welch, S. and Dunlow, L. D. J. Pharmacol. Exp. Ther., 267:390-399, 1993; Rodrigues, A. R A. and Duarte I. D. G. Br. J. Pharmacol, 129:110-114, 2000; Schultz, J. E. J. and Gross, G. J. Pharmacol. Ther., 89:123-137, 2001).
Additionally, several studies have shown that opioid analgesics, including κ opioid agonists affect the mitogen-activated protein kinase (MAPK) (Li, J. G., et al, J. Biol. Chem., 274:12087-12094, 1999; Eitan, S. et al, J. Neuroscience, 23:8360-8369, 2003; Lesscher, H. M. B. et al, Neuroscience, 116:139-144, 2003).
Besides the existence of multiple peptides that present opioid activity, the existence of multiple opioid receptors was pharmacologically characterized. Thus, it is considered that opioid analgesics induce their effects by interaction with specific receptors, constituted of at least 3 main classes: μ (mu), κ (kappa) and δ (delta) (Yaksh, T. L. Eur. J. Anaesthesiol. 1:201-243, 1984), distributed in the Central Nervous System and in peripheral tissues, with distinct pharmacological activities, anatomical distribution and function (Junien, J. L. and Wettstein, J. G. Life Science, 51:2009-2018, 1992; Yaksh, T. L. Acta Anaesth. Scand. 41:94-111, 1997).
The central and peripheral actions of opioids are important components of their therapeutic use. The mu receptors are responsible for most of the analgesic effects of the opioids and for some of their adverse effects, such as respiratory and cardiovascular depression, euphoria, dependence, sedation and alteration of several neuroendocrine functions [Brownstein, M. J. Proc. Natl. Acad. Sci. (USA), 90:5391-5393, 1993].
These secondary effects occur mainly as a consequence of the action of these agonists in the Central Nervous System. This is the main reason for the underuse of opioid analgesics in pain control. Delta opioid receptors are, probably, more important in the periphery, although they also cause central analgesia. In addition to analgesia, these receptors modulate gastrointestinal motility and several hormonal functions. On the other hand, kappa opioid receptors induce analgesia without causing the adverse effects characteristic of μ receptors, such as constipation, itch, respiratory depression, physical dependence and/or addiction. However, the kappa receptors maintain some centrally mediated effects, such as sedation and dysphoria, but not physical dependence (Vanvoigtlander et al., J. Pharmacol. Exp. Ther., 224: 7-12, 1983; Wood, P. L. and Iyengar, S. In: The opioid receptors. Pasternak, G. W. ed. Humana press, Clifton, N.Y., 1988). These receptors are responsible for drinking balance, food intake, intestinal motility, temperature control and several endocrine functions (Leander, J. Pharmacol. Exp. Ther., 227: 35-41, 1983; Leander et al., J. Pharmacol. Exp. Ther. 234, 463-469, 1985; Morley et al., Peptides 4, 797-800, 1983; Manzanares et al., Neuroendocrinology 52, 200-205, 1990; Iyengar et al., J. Pharmacol. Exp. Ther., 238, 429-436, 1986).
Morphine and codeine, the most clinically used opioid analgesics, act as agonists of mu opioid receptors. These opioids cause well-known undesirable adverse effects, for example, the development of physical dependence. Kappa or delta receptor agonists act as analgesics by acting on kappa and delta opioid receptors, respectively. The advantage of these agonists over the classic agonists of mu receptors, e.g morphine, results from their ability to cause analgesia without inducing the undesirable secondary behavioral effects described for morphine. It is known that the structural relationship between opioid receptor and its ligand is responsible for selectivity and specificity for the receptor. Nevertheless, several studies indicate that specific interactions of the opioid receptors with several membrane compartments can contribute to the ability of these opioids to interact selectively with specific receptors (Janecka A. et al. Mini Rev Med Chem. 2:565-572, 2002; Naito A. and Nishimura K. Curr Top Med Chem. 4:135-145, 2004; Singh VK et al. Neuroimmunomodulation. 4:285-297, 1997). This invention refers to novel peptides which are not homologous to the enkephalins, endorphins or dynorphins, and also to the synthetic peptides with preferential activity on kappa opioid receptors.
It is known in the literature that the occurrence of adverse effects using opioids for therapeutic purposes, decreases when opioid specificity and selectivity increases for a specific type or subtype of receptors. Those agonists that have affinity for kappa and/or delta opioid receptors, have demonstrated potent analgesic activity, without presenting serious adverse effects, such as physical dependence, respiratory depression and inhibition of smooth musculature movement, effects that are observed for morphine and agonist derivatives of mu receptors (Nagase, H.; Kawai, K.; Kawamura, K.; Hayakawa, J.; Endoh, T.; patent U.S. Pat. No. 6,323,212, 2001). Adverse effects such as physical dependence and respiratory depression induced by opioids are associated with the action of these drugs on Central Nervous System. The conventional opioids like morphine, naloxone, levorphanol, enkephalins, endorphins and dynorphins and analogs are generally hydrophobic molecules. Therefore, these opioids are able to permeate membranes such as blood-brain barrier, easily accumulating in adipose tissues and organs. This permeability has been also associated with adverse effects in the Central Nervous System, such as euphoria and addiction. Furthermore, these peptides must be administered in high doses, which cause toxic reactions associated with the long exposure to opioids (patent U.S. Pat. No. 5,602,100; Brown, W. L., 1997). Some patents were found in the state of art suggesting the combined use of various antagonists and agonists, formulated or not, as antinociceptive and anti-inflammatory agents. These studies suggest the use of pharmaceutical compositions with concomitant action in different nociceptive pathways and/or inflammatory mechanisms, interfering in the origin of both processes (nociceptive and inflammatory), for example, in surgical processes as oral and/or dental procedures. These agents can be: a 5HT-2 receptor antagonist, a 5HT-3 receptor antagonist, histamine antagonist, serotonin agonist, cyclooxygenase inhibitor, neurokinin 1 receptor antagonist, neurokinin 2 receptor antagonist, purinoreceptors antagonist, calcium channel antagonist, bradykinin B1 receptor antagonist, bradykinin B2 receptor antagonist and a mu opioid receptor agonist. Furthermore, the association of drugs for the treatment of cartilage destruction is also described in these patents and published applications (patent U.S. Pat. No. 6,420,432; Demopulos, G., 2002; US2003096807 A1; Demopulos, G., 2003).
Many works about molecular pharmacology and genetic manipulation of opioid peptides, opioid receptors and opioid receptors agonists and antagonists were found in the state of art. These studies covered the biochemical and molecular effects of opioids, the endogenous opioids neurochemical localization and their behavior-related receptors. Furthermore, the relation of these opioids with analgesia and pain, stress, tolerance and dependence, learning and memory, alcohol and drug abuse, sexual and hormonal activity, pregnancy and endocrine development, general brain activity and locomotion, neurological disorders, gastrointestinal, renal and hepatic functions, and cardiovascular responses were also investigated (Bodnar, R and Hadjimarkou, Peptides, 24, 1241-1302, 2003).
In patent U.S. Pat. No. 5,866,346 (Yu. L., 1999), Lei Yu describes the method of use of dynorphins as ligands for the XOR1 receptor. Therefore, compounds that are preferential kappa opioid receptors agonists, could be ligands for XOR1 receptors.
Although the use of snake venoms and of peptides that act on opioid receptors have been described in the Literature, the nature of the active analgesic substance present in Crotalus durissus terrificus snake venom, or its effectiveness, when administered in the purified form, including oral administration routes, has not been determined yet. Such data also did not explain the effectiveness of compounds analogs to such active substance, nor their specific action on opioid receptors.